Virus assembly is the final step in the life cycle of a virus prior to its release from the infected cell. Enveloped virus assembly is a complex process involving multiple viral and possibly cellular proteins that result in the release of infectious virions by budding at a cellular membrane. Our previous work has provided significant insights into the assembly process of the plus strand RNA enveloped alphaviruses. These viruses have a worldwide distribution and are responsible for severe forms of animal and human disease. Our focus has been in describing the molecular mechanism by which the viral nucleocapsid core is formed. We propose to continue these studies with a set of three specific aims that are directed at: 1) determining the structure and function of the initial building block of the alphavirus nucleocapsid core;2) elaborating the complete pathway for core assembly;and 3) defining the molecular interactions between the E2 glycoprotein and the nucleocapsid core that are responsible for forming an icosahedral enveloped virus. A multi-disciplinary approach will use molecular genetics, biochemistry and structural techniques to probe the mechanism of virus assembly and to ultimately describe the process in atomic detail. Using cryo-electron microscopy image reconstructions of the whole virus, and the atomic structure of the nucleocapsid protein and the E1 and E2 glycoproteins as a guide, site-directed mutagenesis will be carried out to probe structure-function relationships. The resulting mutants will be studied by a variety of in vivo and in vitro biochemical assays that will examine protein-protein and protein-nucleic acid interactions involved in nucleocapsid core formation and interactions of the nucleocapsid core with the icosahedral-organized glycoproteins. Ultimately, understanding this process will aid in the design of novel antivirals and provide a greater appreciation of protein and nucleic acid interactions that are necessary for forming complex macromolecular assemblies.